The goal of this project is to understand the role of protein dynamics and flexibility. NMR will be used to measure the dynamics of mesophilic and hyperthermophilic homologs of adenylate kinase while they are actively turning over, and their flexibility and stability will be compared with their activity at a range of temperatures. Conformational exchange processes on the us-ms timescale will be measured with transverse relaxation experiments as a function of substrate concentration to dissect motions associated with substrate binding and catalysis, and the frequencies of the motions associated with the microscopic steps in the catalytic cycle will be determined using relaxation dispersion methods. Fast motions (ps-ns timescale) which contribute to protein flexibility will also be measured from NMR relaxation data and the temperature dependence of these fast motions will be used to estimate the backbone contribution to thermodynamic properties of the enzyme. The microscopic rates of individual catalytic steps will also be determined by following substrate interconversion with 31P NMR. These experiments will provide quantitative evidence to test the hypothesis that high enzyme activity results from a delicate balance of enzyme stability and flexibility.